It is becoming increasingly clear that the profound dysregulation of Th2 responses underlying allergic inflammation has a strong genetic component. Our group is particularly interested in the impact that natural variation has on the expression and function of key genes that control the differentiation and/or the effector functions of human Th2 cells. GATA-3 acts as a central regulator of murine Th2 responses, and possibly as a bona fide master switch for Th2 cell fate determination. By contrast, very little is known about GATA-3 regulation and function in humans. Numerous GATA-3 single nucleotide polymorphisms (SNP) have been deposited in the existing databases, but no association or functional studies have been reported. We recently identified two SNPs, -7135T/C and -7018C/G, in a region of the human GA TA-3 locus which appears to contain a silencer active in non-T cells. The two SNPs are highly linked; the frequency of the rare - 7018G allele in a large Tucson children population (n=453) was 42%. Notably, homozygosity for -7018G or -7135 C was associated with highly significant increases in the expression of the Th1 cytokine interferon (IFN)-gamma, protection against asthma, and low serum IgE levels. These results suggest that genetic variation in GATA-3 may affect Th2 differentiation and incidence of Th2-mediated disease in vivo. Our long-term goal is to identify the mechanisms whereby genetic variation in GATA-3 modulates the expression of the gene and GATA-3-dependent events. In this application we will test the novel hypothesis that natural variation may affect the epigenetic control of the GATA-3 locus in differentiating CD4+human Th cells. This would result in alterations of GATA-3 expression and GATA-3-dependent events, first and foremost Th2 differentiation and Th2 effector functions. Given the limited time allotted, we will focus exclusively on allele-specific changes in patterns of nuclease hypersensitivity and DNA methylation. Our work will be articulated in three interdependent phases: (1) We will initially use locus-wide Southern blot analysis to map DNase I hypersensitivity and DNA methylation at the GATA-3 locus ( =30 kb) in human CD4+ Th cells isolated from donors homozygous for the GATA-3/-7018 alleles. Cells will be differentiated in vitro under Th1 and Th2 polarizing conditions. Th cell differentiation for each condition will be assessed by measuring expression levels of GATA-3, T-bet, IL-4, IL-13, IL-5, and IFN-gamma, (2) We will then develop high resolution methods to quantify genotype- and/or polarization-specific differences in nuclease hypersensitivity and/or DNA methylation at each site detected by Southern analysis. (3) Finally, we will sequence genomic DNA from -7018/GG and -7018/CC homozygotes encompassing differentially regulated HS and/or DNA methylation sites, in order to merge data from chromatin structure and sequence into a single high resolution map, and correlate variation in chromatin structure with local sequence variation. The functional impact on the epigenetic control of GATA-3 expression of the SNPs thus identified will be analyzed in subsequent studies. The analysis of the relationship between natural variation and epigenetic changes in a critical regulatory gene may establish a novel paradigm and ultimately highlight important mechanisms underlying the pathogenesis of allergic inflammation.